The role of post-translational modifications on NKX6-2 behavior and function

• Autor(a) principal: França, Brenda Gabriella
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/164847
• topic: NKX6-2; Transcription factor; Spastic ataxia 8; Phosphorylation; Transactivation domain; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

NK6 Homeobox 2 (NKX6-2) is a member of the NKX family of transcriptional regulators, responsible for governing oligodendrocyte maturation and the fate of motor neurons and pancreatic cells. Loss-of-function mutations in the NKX6-2 gene result in a rare autosomal recessive genetic disorder known as Spastic Ataxia (SPAX8) with hypomyelinating leukodystrophy. This disease is characterized by cerebellar dysfunction, loss of white matter and, in some cases, atrophy of the vermis and cerebellar hemispheres. Some of the mutations on NKX6-2 affect a putative transactivation domain (TAD), located at the C-terminus of the protein and which function is unclear. Consultation of public databases indicate that TAD contains three residues that have been found phosphorylated in high-throughput mass spectrometry analyses: S246, T252 and Y238. However, the function of these phosphorylation events remains completely unknown. This study attempted to address a fundamental question related to the role of post-translational modifi cations on the intracellular location of NKX6-2. We have developed mammalian expression constructs encoding NKX6-2 fused to the Venus fluorescent reporter, where the transcription factor carries point mutations that inhibit and mimic phosphorylation at S246, T252, and Y238. Specifi cally, mutating S246 and T252 to alanine, and Y238 to phenylalanine, would prevent their phosphorylation while keeping similar structure. On the other hand, mutating S246 and T252 to aspartic acid added an extra negative charge at these sites, partially mimicking phosphorylation. The tyrosine residue was not mutated to mimic phosphorylation, because there are no natural amino acids with an extra negative charge that share the phenolic ring of tyrosine. No mutation produced alterations in the nuclear localization of NKX6-2 in living cells. However, mutations in this protein were associated with nuclear morphology abnormalities, as well as increased protein stability. Additionally, we identified endogenous expression of NKX6-2 in human cancer cell lines where we could study NKX6-2 in normal and pathological, SPAX8- related conditions. These results offer crucial insights into molecular and cellular aspects of NKX6-2 biology, with potential implications for SPAX8.